Journal: The Journal of Experimental Medicine

Article Title: Kallikrein 5 induces atopic dermatitis–like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome

doi: 10.1084/jem.20082242

Figure Lengend Snippet: KLK5 up-regulates TSLP and proinflammatory molecules in keratinocytes. (A) NHKs were stimulated with 400 nM of recombinant KLK5 for 3 h. TSLP , ICAM1 , IL8 , and TNF -α transcripts are increased by seven-, five-, four-, and fivefold, respectively. No difference was observed for IL-1β , TARC , and MDC ( n = 5). (B) NHK transfected with 60 pmol PAR2 siRNA and stimulated with KLK5 showed PAR2 mRNA down-regulation (by 56%) 4 d after transfection. In PAR2 siRNA-transfected cells, KLK5 is less efficient in inducing TSLP , ICAM1 , IL8 , and TNF-α mRNA (48, 55, 60, and 62% of control, respectively; n = 4). (C) In cells treated with 10 µM NF-κB pathway inhibitor (BAY11-7082), KLK5 efficiency to up-regulate TSLP , ICAM1 , IL8 , and TNF-α mRNA is decreased by 81, 70, 59, and 40%, respectively ( n = 3). For each experiment, the mRNAs of interest were measured by quantitative RT-PCR. Each point is the mean of triplicate amplification for at least three independent experiments ( n ). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Cells were transfected with 60 pmol of human PAR2 siRNA or 20 pmol of human KLK5 siRNA (Santa Cruz Biotechnology, Inc.) using the transfection reagent jetSI-ENDO (Ozyme) in serum-free and antibiotic-free EpiLife.

Techniques: Recombinant, Transfection, Control, Quantitative RT-PCR, Amplification

Journal: The Journal of Experimental Medicine

Article Title: Kallikrein 5 induces atopic dermatitis–like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome

doi: 10.1084/jem.20082242

Figure Lengend Snippet: TSLP and proinflammatory molecules are induced by KLK5 in keratinocytes of NS patients. (A) Quantitative RT-PCR on cultured primary keratinocytes reveals that TSLP is significantly overexpressed by fivefold in three NS patients (NSK) compared with three healthy controls (NHK). ICAM1 , IL8 , TNF-α , TARC , and MDC mRNA are overexpressed in two out of three NSKs but not IL-1β mRNA. Each point represents the mean of two independent experiments for each individual. *, P < 0.05. (B) NHK and NSK were treated with 10 µg/ml brefeldin A, and TSLP was detected by immunofluorescence (green). Blue staining is DAPI. Pictures are representative of two independent brefeldin A treatments, each realized on two NHKs and two NSKs. (C and D) NSKs were transfected with KLK5 siRNA and its efficient down-regulation was confirmed by quantitative RT-PCR, Western blotting (laminin 5 used as loading control), and ELISA, 48 and 72 h after transfection. Results are representative of three independent experiments. (E) KLK5 knockdown induces a decrease of TSLP measured by quantitative RT-PCR. ICAM1 , IL8 , and TNF-α mRNAs were less efficiently reduced. Data are the mean ± SD of one experiment, which is representative of three independent experiments. Bars, 49 µm

Article Snippet: Cells were transfected with 60 pmol of human PAR2 siRNA or 20 pmol of human KLK5 siRNA (Santa Cruz Biotechnology, Inc.) using the transfection reagent jetSI-ENDO (Ozyme) in serum-free and antibiotic-free EpiLife.

Techniques: Quantitative RT-PCR, Cell Culture, Immunofluorescence, Staining, Transfection, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: The Journal of Experimental Medicine

Article Title: Kallikrein 5 induces atopic dermatitis–like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome

doi: 10.1084/jem.20082242

Figure Lengend Snippet: KLK5 triggers proinflammatory and proallergic microenvironment independently of external stimuli. Lack of KLK5 inhibition by LEKTI initiates proinflammatory and proallergic cascades independently of environmental factors. In LEKTI-deficient keratinocytes, hyperactive KLK5 directly induces TSLP, IL8, and TNF-α overexpression through PAR2 and NF-κB pathway activation. Unregulated KLK5 also degrades desmosomes at the interface between the GR and the SC leading to defective SC adhesion and IL-1β, IL8, and TNF-α secretion by mechanically stressed keratinocytes. In addition, all these cytokines could induce TARC and MDC chemokines secretion by keratinocytes and dermal fibroblasts. These proinflammatory mediators trigger eosinophilic and mast cell recruitment and activation. TSLP has been reported to activate resident LCs, which migrate to draining lymph nodes and promote the differentiation of naive T cells (Th0) into Th2 cells. Collectively, activated keratinocytes together with eosinophilic and mast cells induce pro-Th2 microenvironment favoring the development of an AD-like phenotype.

Article Snippet: Cells were transfected with 60 pmol of human PAR2 siRNA or 20 pmol of human KLK5 siRNA (Santa Cruz Biotechnology, Inc.) using the transfection reagent jetSI-ENDO (Ozyme) in serum-free and antibiotic-free EpiLife.

Techniques: Inhibition, Over Expression, Activation Assay

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: Lab on a Chip

Article Title: A liver microphysiological system of tumor cell dormancy and inflammatory responsiveness is affected by scaffold properties

doi: 10.1039/C6LC01171C

Figure Lengend Snippet: Fig. 2 Hydrogel scaffolds created a healthier physiological microenvironment and enhanced the responsiveness of hepatic tissue. (A and B) Levels of secreted analytes (pg mL−1) in day 15 effluent from hepatic tissue supported by either polystyrene or hydrogel scaffolds were measured; 53 analytes were above the LoD (n = 5 donors). (A) Scatter plot depicts levels of secreted analytes in hepatic tissue supported by polystyrene vs. hydrogel scaffolds. Significantly altered analytes (black circles) via non-parametric pairwise Wilcoxon rank sum tests (p < 0.05): IL-12p70, IL-13, IL-15, SDF-1α/β and VEGF-A, and those with a >1.5 ratio change (red circles). (B) Graphs summarize analytes exhibiting marked ratio changes >1.5 in polystyrene (top) or hydrogel (bottom) scaffolds. (C) Immunofluorescent images of hepatic tissue formed in polystyrene or hydrogel scaffolds on day 15, obtained using a 20× objective. Red – α-SMA; blue – DAPI. (D and E) Fold changes in acute phase protein production (A1AT and fibrinogen) fol- lowing stimulation with LPS. Protein levels were measured by ELISA on day 15 following stimulation with 1 μg mL−1 LPS for 24 hours (n = 2 do- nors each in duplicate). Fold change was determined by for each pa- tient donor by normalizing each stimulated group to its respective unstimulated control group. Significant differences were determined via non-parametric pairwise Wilcoxon rank sum tests (*p < 0.05).

Article Snippet: Alpha-1-antitrypsin (A1AT) and fibrinogen secretion from hepatic tissue was measured using Enzyme-linked immunosorbent assays (ELISAs) kits according to the manufacturer's instructions (Genway Biotech Inc, San Diego, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Human Mutation

Article Title: A Novel Missense Variant in Ultrarare SLC35A1-CDG Alters Cellular Glycosylation, Lipid, and Energy Metabolism Without Affecting CDG Serum Markers

doi: 10.1155/humu/6290620

Figure Lengend Snippet: Effect of p.Thr45Ala on SLC35A1 expression and transporter activity. (a) Illustration of the cellular route of Neu5Ac. CMP-Neu5Ac is generated by the bifunctional UDP- N -acetylglucosamine 2-epimerase/ N -acetylmannosamine kinase (GNE) converting UDP-GlcNAc to ManNAc and then to ManNAc-6-phosphate. Next, sialic acid synthase (NANS) and N -acetylneuraminate-9-phosphatase (NANP) convert ManNAc-6-phosphate to Neu5Ac via Neu5Ac-9-phosphate. The final step of CMP-Neu5Ac synthesis occurs in the nucleus, where Neu5Ac is linked to the nucleotide CMP by cytidine monophosphate N -acetylneuraminic acid synthetase (CMAS). CMP-Neu5Ac can also be generated through an alternative synthesis pathway, where sialic acid molecules that have been cleaved from glycoconjugates by the lysosomal neuraminidases NEU1-4 are recycled and subsequently reactivated with CMP in the nucleus. (b) Top: variant c.133A>G of Patient 6 localizes to Exon 2 of the SLC35A1 gene. Bottom: electropherogram (Sanger sequencing) of Exon 2 showing homozygosity for variant c.133A>G; p.Thr45Ala in fibroblasts of Patient 6. (c) Transcript and protein expression determined by qPCR and SLC35A1-ELISA revealed a normal mRNA level but significantly reduced SLC35A1 protein level in the patient's cells. (d) Measurement of the import activity of radioactively labelled sugar substrates into Golgi vesicles isolated from control and patient fibroblasts and normalisation of the CMP-[ 14 C]-Neu5Ac import to the import of control sugars GDP-[ 14 C]-fucose and UDP-[ 14 C]-galactose. (e) Protein structure analysis. Left: visualization was performed by PROTTER (Version 1.0; http://wlab.ethz.ch/protter/start/ ). Highlighted in different colours are variants of the six known SLC35A1-CDG patients and of amino acid residues which are important for substrate binding and activity. Threonine 45 (blue circle) of Patient 6 is predicted to be located at the border of the first luminal loop to the second transmembrane domain. Right: due to the p.Thr45Ala variant (Patient 6), an interhelical H-bond is predicted to get lost. Simulation based on the murine Slc35a1 structure generated by DynaMut (PDB: 6XBO). (f) Cycloheximide (CHX) treatment of SLC35A1 K.o. HEK293 cells expressing the wild-type (WT) or the mutated (p.Thr45Ala) SLC35A1-HA protein revealed a shorter half-life of the mutated SLC35A1. The relative amount of SLC35A1-HA was determined by quantification of the Western blot signals where the start of treatment was set to 100%. The regression lines were used to calculate the protein half-life ( y = 50%).

Article Snippet: The SLC35A1 protein level was determined by the usage of the human CMP-sialic acid transporter (SLC35A1) ELISA kit (Abbexa).

Techniques: Expressing, Activity Assay, Generated, Variant Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Isolation, Control, Binding Assay, Western Blot

Journal: Human Mutation

Article Title: A Novel Missense Variant in Ultrarare SLC35A1-CDG Alters Cellular Glycosylation, Lipid, and Energy Metabolism Without Affecting CDG Serum Markers

doi: 10.1155/humu/6290620

Figure Lengend Snippet: Effect of SLC35A1 deficiency on glycosylation in fibroblasts. (a) Lectin binding studies showed a significant increase of nonsialylated structures (RCAI) and a significant decrease in α -2,3 sialylated glycans (MAL-I) in the patient, whereas the amount of α -2,6 sialylated residues (SNA) in SLC35A1-CDG was comparable to controls. (b) Expression of ICAM1 and GP130 is significantly reduced in the patient's cells (Pat.) compared to controls (Ctrl.). For quantification, the fully glycosylated protein forms (glyco., black arrows) were compared to the hypoglycosylated forms (nonglyco, grey arrows). (c) LC-MS analysis of N-glycans in fibroblasts revealed a deviating amount of individual N-glycans in the patient's cells (indicated by black boxes and arrows).

Article Snippet: The SLC35A1 protein level was determined by the usage of the human CMP-sialic acid transporter (SLC35A1) ELISA kit (Abbexa).

Techniques: Glycoproteomics, Binding Assay, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: Human Mutation

Article Title: A Novel Missense Variant in Ultrarare SLC35A1-CDG Alters Cellular Glycosylation, Lipid, and Energy Metabolism Without Affecting CDG Serum Markers

doi: 10.1155/humu/6290620

Figure Lengend Snippet: Metabolic measurements and GlcNAc supplementation assay in fibroblasts. (a) Measurement of very long-chain fatty acids (VLCFAs) in the lysate of patient fibroblasts normalized to the mean values of the control cell lysate revealed partially increased levels. (b) Staining of lipids in control and patient fibroblasts with Oil Red O presented with an increase in pronounced coloration in the case of the patient's cells indicating an accumulation of neutral lipids. (c) Nanoelectrospray ionization tandem mass spectrometry of cellular lipids revealed significant alterations concerning ceramide (Cer), phosphatidylethanolamine plasmalogen (PE-P), and triacylglycerol (TAG) within the main lipid classes. Abbreviations: CE, cholesteryl ester; Chol, cholesterol; DAG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; Hex2Cer, dihexosylceramide; HexCer, hexosylceramide; LPC, lysophosphatidylcholine; PA, phosphatidic acid; PG, phosphatidylglycerol. (d) Reduced expression of ATP5H combined with a decreased ATP level (e) hint to a general mitochondrial impairment in SLC35A1-CDG. (f) Reduced proliferation due to decreased growth rate and increased doubling time in the patient's cells can be improved by 10 mM GlcNAc as a cell culture supplement.

Article Snippet: The SLC35A1 protein level was determined by the usage of the human CMP-sialic acid transporter (SLC35A1) ELISA kit (Abbexa).

Techniques: Control, Staining, Mass Spectrometry, Expressing, Cell Culture